Structural Study of ARF-GTPase Regulators and Effectors

ARF-GTPase 调节器和效应器的结构研究

• 批准号: 7255580
• 负责人: JONATHAN D GOLDBERG
• 金额: $ 29.43万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7255580
• 关键词: ARFGAP1; Acceleration; Active Sites; Aluminum; Anodes; Binding; Biochemical; Biogenesis; Brefeldin A; Capsid Proteins; Cells; Coat Protein Complex I; Coated vesicle; Complement; Complex; Cytosol; Dissection; Eukaryotic Cell; Exocytosis; Family; GTP Binding; GTPase-Activating Proteins; Goals; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hand; Homologous Gene; Housing; Hydrolysis; Insecta; Investigation; Kinetics; Localized; Maintenance; Membrane; Molecular; Molecular Conformation; Mycotoxins; Neurotransmitters; Nucleotides; Organelles; Phase; Process; Property; Proteins; Protocols documentation; Publishing; Purpose; Range; Rate; Reaction; Research; Resolution; Roentgen Rays; Role; Site; Source; Staging; Structure; System; Testing; Transport Vesicles; Vesicle; Work; analog; base; beryllium fluoride; catalyst; conformational conversion; improved; insight; interest; member; mouse Gdi2 protein; programs; protein expression; release factor; uptake

DESCRIPTION (provided by applicant): ARF GTPases are key regulators of vesicle transport steps and thereby control a broad range of processes in eukaryotic cells, including the maintenance of intracellular compartments and the exocytosis of proteins and neurotransmitters. Research in this proposal focuses on an integrated understanding of ARF function in the processes of vesicular coat assembly and regulated disassembly. Vesicle assembly is triggered by the GTP-dependent recruitment of vesicular coat complexes by ARF. We will explore the mechanism of GDP-to-GTP conversion on ARF1 by determining the crystal structure of a long-sought-after intermediate in the nucleotide exchange reaction: ARF1-GDP complexed with its Sec7 catalyst, trapped using the fungal toxin, Brefeldin: high-quality crystals (2.4 Angstroms resolution) are in hand. We will explore the process of coat recruitment through analyses of ARF-coat interactions, involving the biochemical dissection of heptameric (550 kDa) COPI and tetrameric (400 kDa) COPII coats. For COPI, we have established protein expression systems for six subcomplexes, and plan now to determine the structure of ARF1 bound to an "active" COPI subcomplex. For the COPII coat, we previously determined the crystal structure of the inner shell of the coat bound to the ARF homolog, Sar1. We have now dissected the COPII outer shell biochemically, and will determine the crystal structure of a core outer-shell complex: high-quality crystals (2.6 Angstroms resolution) are in hand. Finally, we will explore vesicular coat disassembly by focussing on the GTP hydrolysis reaction that triggers this process. We previously discovered that COPI accelerates GTP hydrolysis in the ARF1-ARFGAP complex by 300-fold or more. We will investigate the basis for this effect by biochemical and crystallographic analysis of COPI core bound to ARF1-ARFGAP in ground- and transition-state complexes. In the COPII system, maximal rate acceleration of the GTPase requires catalytic contributions from both the inner and outer shells of the coat. We recently defined an assembly capable of maximal rate acceleration that includes inner (Sec23) and outer (Sec31 fragment) shell molecules complexed with Sar1 GTPase. We have obtained small crystals of this complex that diffract to at least 2.8 Angstroms resolution in-house, which we are working to improve. These studies will provide insight to the processes of GTP-dependent assembly of vesicular coat complexes and coat-controlled GTP hydrolysis triggering disassembly.

描述（由申请人提供）：ARF GTP酶是囊泡运输步骤的关键调节剂，从而控制真核细胞中的广泛过程，包括细胞内区室的维持以及蛋白质和神经递质的胞吐作用。本提案的研究重点是对囊泡外壳组装和调节拆卸过程中 ARF 功能的综合理解。囊泡组装是由 ARF 依赖 GTP 募集囊泡外壳复合物触发的。我们将通过确定核苷酸交换反应中长期寻找的中间体的晶体结构来探索 ARF1 上 GDP 到 GTP 转化的机制：ARF1-GDP 与其 Sec7 催化剂复合，并使用真菌毒素 Brefeldin 捕获：高品质晶体（2.4埃分辨率）已在手。我们将通过分析 ARF-涂层相互作用来探索涂层招募过程，包括七聚体 (550 kDa) COPI 和四聚体 (400 kDa) COPII 涂层的生化解剖。对于COPI，我们已经建立了六个亚复合物的蛋白质表达系统，现在计划确定与“活性”COPI亚复合物结合的ARF1的结构。对于 COPII 涂层，我们之前确定了与 ARF 同系物 Sar1 结合的涂层内壳的晶体结构。我们现在已经对 COPII 外壳进行了生化解剖，并将确定核心外壳复合物的晶体结构：高质量晶体（2.6 埃分辨率）已在手。最后，我们将重点关注触发这一过程的 GTP 水解反应，探索囊泡层的分解。我们之前发现 COPI 可将 ARF1-ARFGAP 复合物中的 GTP 水解加速 300 倍或更多。我们将通过对基态和过渡态复合物中与 ARF1-ARFGAP 结合的 COPI 核心进行生化和晶体学分析来研究这种效应的基础。在 COPII 系统中，GTPase 的最大速率加速需要涂层内壳和外壳的催化贡献。我们最近定义了一种能够实现最大速率加速的组件，其中包括与 Sar1 GTPase 复合的内壳分子（Sec23）和外壳分子（Sec31 片段）。我们已经获得了这种复合物的小晶体，其内部衍射分辨率至少为 2.8 埃，我们正在努力改进这一点。这些研究将深入了解囊泡外壳复合物的 GTP 依赖性组装过程以及外壳控制的 GTP 水解引发的分解过程。